Microscopy is an essential tool that is driving progress in cell biology, and is still the only practical means of obtaining spatial and temporal resolution for living cells and tissues observation. Microscopy involves electromagnetic radiation transmitted through or reflected from the sample through a single or multiple lens element(s) to allow a magnified view of the sample. The resulting image can be detected directly by the eye, imaged on a photographic plate or captured digitally.
Over the centuries, optical microscopy has addressed the need to see what happens at the microscopic level. As the needs evolved, so did microscopy. This way, higher magnification objectives and aberration-corrected objectives respectively answered the need to produce larger images and images of higher quality, while better resolved objectives and shorter wavelength sources answered the need to see and distinguish smaller and smaller elements.
But as the investigated objects became more and more complex, the need to have better and even material-specific contrast arose. Fluorescence, phase contrast and nonlinear microscopy were thus developed.
Fluorescence Microscopy
The most commonly encountered solution for material-specific contrast is fluorescence microscopy. Fluorescence is the emission of electromagnetic radiation by a substance that has beforehand absorbed electromagnetic radiation of a different wavelength. In fluorescence microscopy, the electromagnetic radiation resulting from fluorescence is separated from the excitation by some technical mean, and contributes alone to the image formation.
The fluorescent substance can be the material of interest, but most generally it consists in a marker, called fluorophore, biochemically functionalized to bind to the material of interest. Using such exogenous marker is referred to as staining and is not always ideal, as the marker can affect the object and alter its behavior or characteristics. Fluorophores can even be toxic to the object of interest. Although exogenous labels are generally applicable to any animal model and benefit from decades of acceptance in the laboratory, there has been a recent trend toward imaging with genetically encoded markers, mostly green fluorescent proteins (GFPs) or their variants. Because they can be encoded in DNA, these endogenous genetic markers can be globally targeted to well-defined regions in intact animals.
Unfortunately, fluorophores lose their ability to fluoresce as they are illuminated in a process called photobleaching. Special care must be taken to prevent photobleaching through the use of more robust fluorophores, by minimizing illumination, or by introducing a scavenger system to reduce the rate of photobleaching.
Phase-contrast Imaging
In phase contrast microscopy, small phase shifts in the light passing through a transparent object are converted into amplitude or contrast changes in the image. A phase contrast microscope does not require staining to view the slide. Nowadays, there exist many techniques for phase-contrast imaging.
Among the most interesting are the techniques—often based on interferometric (or holographic) principles—that provide quantitative phase imaging. These techniques literally added a new dimension to microscopy by making possible nanometer-scale surface measurements, and very precise refractive index tomography. Digital holographic microscopy (DHM) is one of such techniques [E. Cuche, P. Marquet and C. Depeursinge, “Simultaneous amplitude-contrast and quantitative phase-contrast microscopy by numerical reconstruction of Fresnel off-axis holograms,” Applied Optics, 38. p. 6994-7001 (1999)].
Nonlinear Microscopy
Nonlinear optics is a relatively new trend in microscopy. The main idea of this technique is to exploit the nonlinear responses of the polarization of material to electric field to generate nonlinear radiations that will form images. The differences between the nonlinear responses of different materials provide a highly specific contrast.
As the probability for nonlinear processes to occur is very low, the excitation electromagnetic source, generally a femtosecond laser, is tightly focused in the object. As a consequence, background signal is strongly suppressed, since the probability that multiphoton processes occur outside the focus volume of the excitation beam is negligible.
Furthermore, as the generated signal generally lies in the visible or near-infrared region of the electromagnetic spectrum, the excitation source is generally a near-infrared radiation. Near-infrared radiation is much less absorbed and scattered by biological tissues and makes possible deep tissue imaging. In addition, these lower-energy photons are less likely to cause damage outside the focal volume.
Nonlinear microscopy generally uses a scanning, confocal-type microscope. Because they require scanning of the illumination or the object, such microscopes are intrinsically slow and vulnerable to vibrations.
Nonlinear microscopy can be divided in two distinct categories: incoherent and coherent nonlinear microscopy.
Incoherent Nonlinear Microscopy
Incoherent nonlinear microscopy produces signals whose phase is random and whose power is proportional to the concentration of radiating molecules.
Multiphoton fluorescence microscopy, a fluorescence imaging technique that allows imaging of living tissue up to a much higher depths [W. Denk, and K. Svoboda, “Photon upmanship: Why multiphoton imaging is more than a gimmick,” Neuron, 18. p. 351-357 (1997)], is an example of incoherent nonlinear microscopy. Multiphoton fluorescence microscopy requires multiple photons to be absorbed simultaneously to provide enough energy to generate free charge carriers in the material. This special case differentiates from the above-mentioned fluorescence microscopy by having emitted EM radiation with a shorter wavelength (and higher energy) than the absorbed EM radiation and requiring multiple excitation photons to be absorbed simultaneously.
Even though some materials have intrinsic fluorescence properties, multiphoton fluorescent microscopy relies on markers, or fluorophores, to provide image contrast.
Coherent Nonlinear Microscopy
Coherent nonlinear microscopy relies on signals whose phase is rigorously prescribed by a variety of factors, including the excitation light phase and the geometric distribution of the radiating molecules. Coherent signal power is proportional to the concentration of radiating molecules squared. Nonlinear coherent microscopy is based on the simultaneous scattering of two or more photons. Its main advantage over fluorescence lies in the fact that nonlinear interactions occur instantaneously and theoretically make possible ultrafast measurements.
Harmonic generation, coherent anti-Stokes Raman scattering, sum- and difference-frequency generation are examples of coherent nonlinear microscopy.
Nonlinear microscopy can rely on markers to provide contrast, but does not exclusively require so. Indeed, most materials have intrinsic nonlinear response of some sort, or lack of, which provide contrast to nonlinear microscopy. Using intrinsic nonlinear properties of materials reduces the amount of time and efforts for sample preparation and avoids its contamination by possibly toxic, or chemically active, markers. Nevertheless, contrast agents can still be used for their nonlinear responses and high selectivity, especially when functionalized, or bioconjugated. One example is the use of styryl dye derivatives as an effective Second Harmonic Generator (SHG) sensor of membrane potential [A. C. Millard, L. Jin, M.-D. Wei, J. P. Wuskell, A. Lewis, L. M. Loew, “Sensitivity of second harmonic generation from styryl dyes to transmembrane potential,” Biophys J 86. p. 1169-1176 (2004)]. It is an emerging trend to develop markers specifically for coherent nonlinear microscopy applications [C. L. Hsieh, R. Grange, Y. Pu and D. Psaltis, “Bioconjugation of barium titanate nanocrystals with immunoglobulin G antibody for second harmonic radiation imaging probes,” Biomaterials 31. p. 2272-2277 (2010), J. Extermann, L. Bonacina, E. Cuna, C. Kasparian, Y. Mugnier, T. Feurer and J. P. Wolf, “Nanodoublers as deep imaging markers for multi-photon microscopy,” Optics Express 17. p. 15342-15349 (2009).]. A large variety of nanocrystals, among which are BaTiO3. ZnO, KTiOPO4 (KTP), Fe(IO3)3 and KNbO3. were thus developed for harmonic generation imaging.
Gold nanoparticles are especially promising as nonlinear markers for biological. First, they are totally biocompatible, chemically inert and non-toxic. In addition, a vast knowledge of surface chemistry of noble metals has already been acquired, which makes their functionalization, or bioconjugation, relatively easy. Finally, resonance effects, such as surface plasmon resonance, provide tremendous signal enhancement factors.
Nonlinear Holography
Nonlinear holography, an emerging microscopy technique, consist in exploiting the coherent nature of nonlinear interactions to record the interference between the nonlinear wave generated by the object and a nonlinear reference wave of the same nature.
Intensity images are obtained from processing of the recorded interference patterns, and a single hologram contains all the information for a 3D tomography of the nonlinear EM wave intensity. A direct consequence of this is that nonlinear holography requires no scanning of any sort. In particular, it makes nonlinear holography vibration-insensitive.
Nonlinear harmonic holography [PCT applic.number PCT/US07/85409] has been described as a technique and system that combines holography and the nonlinear interaction named second harmonic generation, and that enables holographic recording of 3D intensity images with femtosecond framing time.
Nonlinear Phase-contrast Imaging
Just as phase imaging added a new dimension to classical (or linear) optical microscopy, nonlinear phase imaging is expected to provide additional information, inaccessible to state of the art nonlinear imaging techniques, and thus open a large panel of new applications. Microscopy techniques capable of recovering the phase of nonlinear electromagnetic waves will become of great interest.